"Mr. President, we urge you to bring a renewed sense of urgency to the process," said the letter, publicized today by authors Robert Menendez, the Democratic chairman of the Senate Foreign Relations Committee, and Republican Senator Lindsey Graham.
"We need to understand quickly whether Tehran is at last ready to negotiate seriously" on its nuclear program, it said.
Another 74 senators signed the letter, which was sent to the White House on Friday, two days before Rowhani was sworn in in Tehran.
Despite Iranian denials, the West is convinced Tehran is pursuing a nuclear bomb.
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"Iran has used negotiations in the past to stall for time, and in any event, (supreme leader Ayatollah Ali) Khamenei is the ultimate decision-maker for Iran's nuclear program," the senators wrote to Obama.
"Iran needs to understand that the time for diplomacy is nearing its end. We implore you to demand immediate serious moves on Iran's part."
Last week shortly before Congress went on summer break, the House of Representatives overwhelmingly approved harsh new sanctions aimed at drastically reducing Iran's oil exports by ending purchasing exemptions to countries like China and Japan.
The recess window is being seen as a pivotal period for both the White House and Iran.
A Senate official told AFP that after the August recess the Senate Banking Committee aims to introduce legislation on further sanctions against the Islamic republic.
"We must be prepared to act, and Iran must see that we are prepared.